Hi all, I realise this is probably asked quite a bit so I apologise in advance!

I am looking to trouble shoot my competent cell making process. I make competent cells for a number of labs, but struggle to get the transformation efficiency of chemically competent cells above 10^7 (I get say 2 x 20^7 at best). I am very careful to ensure everything is cold. Any help here would be appreciated.

I've pasted the exact method I use below this:

• Pre-chill a flask on ice for the chilling step• Pre-chill the centrifuge and its rotor• Work in the cold room!• Pre-chill eppendorfs in the -70 freezer, and tips in the cold room, (and maybe an eppendorf rack in the -80)

1. Make a 5ml pre-culture overnight from a single colony, in SOC medium2. The next day, inoculate 100ml pre-warmed SOC medium with 5ml of the overnight pre-culture. The volume of the flask here is important for aeration. I use a 2 litre baffled flask for improved aeration (volume of medium 1/10 or 1/30 volume of the flask).3. Incubate the culture at 37°C with vigorous shaking until the OD600 reaches 0.4-0.6 (4-7x107 cells/ml). 4. When at the correct OD, chill the culture on ice for 10-15 minutes. I use a pre-chilled flask for this in a wet ice bath.5. Spin the culture at 3000 RPM for 15 minutes at 4°C in a pre-chilled rotor.6. Resuspend the pellet in RF1 solution. The volume should be 1/3 of the culture volume7. Incubate the cell suspension on ice for 15 minutes.8. Pellet the cells as in step 5.9. Resuspend the pellet in 8ml (1/12.5 of the original culture volume) of RF2.10. Incubate the cell suspension on ice for 15 minutes.11. Aliquot the cells into pre-chilled eppendorfs.12. (Immediately flash freeze the aliquots in liquid nitrogen, then store at -70°C.)